PROJECT SUMMARY (Project 3; Klimecki) Arsenic is one of the most common metals found as a contaminant at Superfund sites, particularly those involving legacy mining operations. Arsenic may be the most striking example of an incontrovertible human carcinogen for which there is little scientific agreement as to mechanism of action. We recently published a novel effect of inorganic arsenic in cultured human cells, the shift from oxidative energy metabolism to anaerobic glycolysis, in what has the appearance of an inappropriately launched hypoxia response. The scientific literature is witnessing exponential growth in reports linking fundamental energy metabolism to the development and progression of cancer. We believe that the disruption of energy metabolism, a process occurring in all cells of the body, could provide a rational explanation for the remarkable diversity of arsenic targets of carcinogenicity-the disruption of a fundamental process that manifests as a similar disease in multiple tissue contexts. Preliminary data that we present in this proposal demonstrates that genetically manipulating the hypoxia-mimetic effect of arsenite in an in vitro model of arsenite-induced lung cancer has a significant impact on arsenic carcinogenicity. Overall Objective: The objective of this superfund research project is to accurately define the extent of the perturbation of cellular energy metabolism by arsenic, and to identify the important regulators of this process. Finally we aim to establish the in vivo correlates of this effect while testing the novel hypothesis that arsenite could lead to malignancy not only by its effect on cells destined to become malignant, but also by exerting its hypoxia-mimetic effect on surrounding stromal cells. Building on a single published study in 2005 that observed this type of stromal enhancement of tumor growth in mice, successful conformation and further dissection of this effect would be a paradigm shifting finding. Hypothesis: The carcinogenic mechanism of inorganic arsenic involves its hypoxia-mimetic response, and includes HIF- mediated pathway gene induction, as well as altered carbohydrate metabolism that favors the production of macromolecular precursors. This effect of arsenic facilitates malignancy in both tumor cells as well as surrounding stroma. We will structure this effort through three Aims: (1) Define the temporal order and occurrence of metabolic disruption, loss of epithelial identity, and malignant transformation in a non-malignant pulmonary epithelial cell line, BEAS-2B; (2) Define key components of the arsenite-induced ?pseudo-hypoxic? response and their contribution to early events leading to malignant transformation; and (3) Establish in vivo correlates of the hypoxia mimetic effect of arsenite on malignancy.